1. Field of the Invention
The present disclosure relates to the isolation, maintenance and propagation of human embryonic stem cells (hESC) from the inner cell mass of surplus embryos. This disclosure also relates to the characterization of isolated human ES cell lines, thereby demonstrating their in vitro differentiation potential and their prospective use in cell therapy and drug screening.
2. Description of Related Art
Pluripotent stem cells that are derived from the inner cell mass of a blastocyst are referred to as embryonic stem cells, while stem cells derived from primordial germ cells of the developing gonadal ridge are referred to as embryonic germ cells (Shamblott et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95(23):13726-31). Embryonic stem (ES) cells have been derived from the inner cell mass (ICM) of mammalian blastocysts (Evans and Kaufman, (1981) Nature, 292(5819):151-9; Martin, (1981) Proc. Natl. Acad. Sci. U.S.A., 78:7634-8). These cells are pluripotent, and are capable of developing into any organ or tissue type. ES cells are capable of proliferating in vitro in an undifferentiated state, maintaining a normal karyotype through prolonged culture, and maintaining the potential to differentiate into derivatives of all three embryonic germ layers (i.e., mesoderm, ectoderm and endoderm) (Itskovitz-Eldor et al., (2000) Mol. Med., 6(2):88-95).
ES cells represent a powerful model system for the investigation of mechanisms underlying pluripotent cell biology and differentiation within the early embryo, as well as providing opportunities for genetic manipulation. Appropriate proliferation and differentiation of ES cells can be used to generate an unlimited source of cells, suitable for cell-based therapies of diseases that result from cell damage or dysfunction.
ES cells have been isolated from the ICM of blastocyst-stage embryos in mice (Solter and Knowles, (1975) 72(12):5099-5102), as well as several other species. For example, pluripotent cell lines have also been derived from pre-implantation embryos of several domestic and laboratory animal species, such as bovine (Evans et al., (1990), Theriogenology, 33:125-8), porcine (Evans et al., (1990) supra; Notarianni et al., (1990) J. Reprod. Fertil. Suppl., 41:51-6), sheep and goat (Meinecke-Tillmann and Meinecke, (1996), J. Animal Breeding and Genetics, 113:413-26; Notarianni, et al., (1991), J. Reprod. Fertil. Suppl., 43:255-60) rabbit (Giles et al., (1993) Mol. Reprod. Dev., 36(2):130-8; Graves et al., (1993) Mol. Reprod. and Dev., 36:424-33), mink (Sukoyan et al., (1992), Mol. Reprod. and Dev., 33:418-31), rat (Iannaccona et al., (1994), Dev. Biology, 163:288-92), hamster (Doetschman et al., (1985) J. Embryol. Exp. Morphol., 87:27-45), and rhesus and marmoset monkeys (Thomson et al., (1995) Proc. Natl. Acad. Sci. 92(17):7844-8; and Thomson, et al., (1996), Biol. Reprod., 55:254-59). Thomson et al. (1998) Science 282(5391):1145-7 and Reubinoff et al. (2000) Nat. Biotech. 18(5):559) have reported the derivation of human ES cell lines.
Early work on ES cells was done in mice (Doetschman et al., (1985) J. Embr. Exp. Morphol., 87:27-45). Mouse ES cells are undifferentiated pluripotent cells derived in vitro from preimplantation embryos, and maintain an undifferentiated state through serial passages when cultured in the presence of fibroblast feeder layers and leukemia inhibitory factor (LIF). Although research with mouse ES cells facilitates the understanding of developmental processes and genetic diseases, significant differences in human and mouse development limit the use of mouse ES cells as a model of human development. The morphology, cell surface markers and growth requirements of ES cells derived from other species are significantly different than for mouse ES cells. Further, mouse and human embryos differ significantly in temporal expression of embryonic genes, such as in the formation of the egg cylinder versus the embryonic disc (Kaufman, The Atlas of Mouse Development; London; Academic Press, 1992), in the proposed derivation of some early lineages (O'Rahilly and Muller; Developmental stages in Human Embryos, Washington; Carnegie Institution of Washington, 1987), in the structure and function of the extraembryonic membranes and placenta (Mossman, Vertebrate Fetal membranes; New Brunswick; Rutgers, 1987), in growth factor requirements for development (e.g., the hematopoietic system-Lapidot Lab. Animal Sciences 1994), and in adult structure and function (e.g., central nervous system). To overcome these differences and to have a better insight into human embryonic development, ES cells were successfully established from primates (Thomson et al., 1995 and 1998, supra).
The cell lines currently available that most closely resemble human ES cells are human embryonic carcinoma (EC) cells, which are pluripotent, immortal cells derived from teratocarcinomas (Andrews et al., (1984) Lab. Invest. 50(2):147-162; Andrews et al., in: Robertson E., ed. Teratocarcinomas and Embryonic Stem Cells: A Practical Approach. Oxford: IRL press, pp. 207-246, 1987). EC cells can be induced to differentiate in culture, and the differentiation is characterized by the loss of specific cell surface markers (SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) and the appearance of new markers (Andrews et al., (1987), supra). Human EC cells will form teratocarcinomas in nude mice with derivatives of multiple embryonic lineages in the tumors. Similar mouse EC cell lines have been derived from teratocarcinomas, and, in general, their developmental potential is much more limited than mouse ES cells (Rossant and Papaioannou, (1984) Cell Differ. 15:155-161). Teratocarcinomas are tumors derived from germ cells, and although germ cells (like ES cells) are theoretically totipotent (i.e., capable of forming all cell types in the body), the more limited developmental potential and the abnormal karyotypes of EC cells are thought to result from selective pressures in the teratocarcinoma tumor environment (Rossant and Papaioannou, (1984), supra). ES cells, on the other hand, are thought to retain greater developmental potential because they are derived from normal ES in vitro, without the selective pressures of the teratocarcinoma environment.
The first human pluripotent ES cell line was published in 1998 (Thomson et al., (1998), supra). A few years later, human embryonic stem cell lines (“human ES cell lines”) were established from human blastocysts (Reubinoff et al., (2000), supra). To date, the majority of described human ES cell lines have been derived from day 5 to day 8 blastocysts produced for clinical purposes after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). In addition, the isolation of ICM from the morula (day 4 embryo) stage has also been reported (Giles et al., 1993).
Human ES cells can be isolated from human blastocysts. Human blastocysts can be obtained from human in vivo pre-implantation embryos or from IVF embryos, intracytoplasmic sperm injection, ooplasm transfer, or other methods well known to those of skill in the art. Human ES cells may be derived from a blastocyst using standard immunosurgery techniques as disclosed in U.S. Pat. Nos. 5,843,780 and 6,200,806, Thomson et al., (1998), supra, and Reubinoff et al., (2000), supra (each incorporated herein by reference), whole embryo-culture method, or by a unique method of laser ablation (U.S. Ser. No. 10/226,711, incorporated herein by reference). Alternatively, a single cell human embryo can be expanded to the blastocyst stage. Although numerous human ES cell lines have been derived to date, only a few of them are well characterized in terms of their unique identity, self-renewal capacity and differentiation potential (Brimble et al., (2004) Stem Cells Dev., 13:585-7).
One method well known to those of skill in the art for generating human ES cells is by immunosurgery. This method involves removing the zona-pellucida from the blastocyst and isolating the ICM by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture flask containing the appropriate medium, which enables its outgrowth. After 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by enzymatic degradation, and the cells are re-plated in a fresh tissue culture medium. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re-plated. Resulting ES cells are then routinely split every 1-2 weeks to maintain the cells in a generally undifferentiated state. For a more detailed description of the immunosurgery technique, see U.S. Pat. No. 5,843,780; Thomson et al., (1998), supra; Thomson et al., (1998) Curr. Top. Dev. Biol. 38:133; Thomson et al., (1995), supra; Bongso et al., (1989) Hum. Reprod. 4(6):706-13; Gardner et al., (1998), Fert. and Sterility, 69(1):84-8), each of which is incorporated herein by reference.
Methods of maintaining human ES cells in an undifferentiated pluripotent state include but are not limited to culturing the cells in the presence of a feeder layer, under feeder-free conditions, in the presence of conditioned medium, and/or on an extra-cellular matrix supplemented with serum or conditioned medium. The feeder layers may be, for example, γ-irradiated or mitomycin-C treated mouse embryonic fibroblast (MEF) cells or human fibroblast cells. When cultured in a standard culture environment in the absence of a feeder layer, human ES cells may rapidly differentiate or fail to survive. Unlike murine ES cells, the presence of exogenously added LIF does not prevent differentiation of human ES cells. Feeder cell layers are used to provide a microenvironment (or niche) to prevent stem cells from differentiating along their natural course. These feeder layers appear to provide the stem cells with external signals such as secretion of factors and cell-to-cell interactions mediated by integral membrane proteins. Watt and Hogan, (2000) Science 287(5457):1427-30. In light of the fact that secretion factors and direct cell-to-cell interactions control in vitro survival, proliferation, and differentiation of the stem cells, an ideal environment should consist of healthy feeder tissues with normal microstructures and functions, or simulate such an environment. Examples of feeder cells include but are not limited to: (1) irradiation-inactivated mouse embryonic fibroblasts; (2) mitotically (mitomycin C) inactivated mouse embryonic fibroblasts; and (3) irradiation-inactivated STO fibroblast feeder layers. See Thomson et al., (1998) supra; Reubinoff et al. (2000), supra; and Shamblott et al., (1998) Proc. Natl. Acad. Sci. U.S.A. 95(23): 13726-31, each incorporated herein by reference.
In spite of the progress in effectively culturing ES cells, several significant disadvantages with these methods still exist. For example, exposure to animal pathogens through MEF-conditioned medium or matrigel matrix is still a possibility. The major obstacle of the use of human ES cells in human therapy is that the originally described methods to propagate human ES cells involve culturing the human ES cells on a layer of feeder cells of non-human origin, and in the presence of nutrient serum of non-human origin. More recently, extensive research into improving culture systems for human ES cells has concentrated on the ability to grow ES cells under serum free/feeder-free conditions. For example, to ensure a feeder-free environment for the growth of human ES cells, a substitute system based on medium supplemented with serum replacement (SR), transforming growth factor β1 (TGF-β1), LIF, bFGF and a fibronectin matrix has also been tried (Amit et al (2004), Biol. Reprod. 70(3):837-45). Evaluation of methods for derivation and propagation of undifferentiated human ES cells on human feeders or feeder-free matrices continues.
Detailed characterization of human ES cells may include analysis at the cellular and molecular level, as well as an analysis of the regulation of cell cycle, expression of high telomerase activity, genetic stability, particular HLA and STR types, and differentiation potential under in vitro and in vivo conditions. The profile of surface antigens displayed in undifferentiated human ES cells matches that of human ES cells and human EC cells. Undifferentiated human ES express globo-series cell surface markers such as stage specific embryonic antigens (SSEAs), for example SSEA-3 and SSEA-4, as well as tumor recognition antigens, for example TRA-1-60 and TRA-1-81. In addition, human ES cells express POU5F1, promoter-encoded transcription factor OCT-4, E-cadherin and the gap junction protein connexin-43 (Andrews et al., 2002). Unlike mouse ES cells, undifferentiated human ES cells do not express SSEA-1. Undifferentiated human ES cells stain positively for alkaline phosphatase, and demonstrate high telomerase activity indicative of their increased capacity for self-renewal.
The genetic stability of human ES cells can be assessed by using the standard G-banding technique, which is well-known to a person of ordinary skill in the art. Normally human ES cells maintain a stable karyotype, either 46 XX or 46 XY, even after prolonged continuous culture. With increased passaging, however, the cells tend to show abnormal karyotypes including trisomies of chromosomes 12-17 and the X chromosome. The unlimited proliferative potential of ES cells is directly correlated with telomerase activity. A Telomerase Repeat Amplification Protocol (TRAP) assay may be performed to assess telomerase activity in a particular ES cell line. The assay may be performed either using a radioisotopic method (Thomson et al., (1998), supra, or a non-radioisotopic method (Oh et al., (2004) Stem Cells 23(2):211-19).
Human ES cells have the potential to differentiate into all cell types of the human body. The developmental potential of these cells after prolonged culture may be examined in vitro through the formation of embryoid bodies and in vivo through the formation of teratomas in SCID mice (Evans and Kaufman, (1983), supra). To confirm that human ES cells retain their in vitro differentiation capacity, embryoid bodies can be formed in suspension culture and analyzed by RT-PCR and immunocytochemistry for markers representing each of the three germ layers (Itskovitz-Eldor, (2000), supra, and Shamblott et al., (1998), supra).
Human ES cells offer insight into developmental events, which cannot be studied in explant systems. Screens based on the in vitro differentiation of human ES cells to specific lineages can identify gene targets, which can be used to design or reprogram tissue generation or regeneration, as well as identify teratogenic or toxic compounds. Replacement of non-functional cells, tissues, or organs using ES cell technology may offer a therapeutic treatment in the case of degenerative diseases like Parkinsons disease, stroke, cardiac ischemia, hepatic failure, juvenile-onset diabetes mellitus, or other diseases or conditions that result from the death or dysfunction of one or several cell types (Wobus and Boheler, (2005), Physiol. Rev. 85(2):635-8). Nevertheless, in order for the potential therapeutic applications of human ES cell technology to become reality, techniques must enable the production of enriched human ES-cell-derived specialized cell types under defined growth conditions, a pathogen-free environment, and survival under extended in vitro conditions.
At present, there are a limited number of human ES cell lines available and they represent a very small sample of the genetic diversity of the human population. Hence, there is an urgent need for the generation and characterization of additional cell lines, as each cell line may have its own set of characteristics and advantages for different applications in a particular population. Furthermore, the availability of more human ES cell lines for comparison will facilitate the global efforts to define the criteria of human ES cells and the establishment of appropriate and robust methods for the maintenance and expansion of human ES cells.